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1 AN INTRAMITOCHONDRIAL POLYPEPTIDE WITH STRONG CHELATING PROPERTIES FOR COPPER: AN AGENT THAT MONITORS CYTOTOXICITY F. STEPHEN VOGEL, M.D., AND LIEsEoLTTE KEMPER From the Departnent of Pathology, Duke University Medical Center, Durham, N.C. In low concentrations, copper uncouples oxidative phosphorylation of mitochondria isolated from the brain tissue of a wide variety of animal species." The present studies broaden this observation, initially providing evidence that the metal regularly reacts in like manner with mitochondria from the myocardium and skeletal muscle of the rat, yet, disparately, not with those from the liver, kidney, spleen or lung of the same animal. Since oxidative phosphorylation is a universal characteristic of this organelle, these findings raise the possibility that certain mitochondria are rendered nonreactive by the presence of a component or components that have the proclivity to inactivate the metal, as by chelation. In development of this possibility, chelating constituents were sought in the splenic mitochondria of the rat; a polypeptide with avid chelating properties for copper was disclosed. Following its extraction and purification, initial efforts were made to characterize its nature. MATERIAL AND METHODS The capacities of mitochondria and mitochondrial extracts to chelate copper were tested in a standardized model, wherein the uncoupling reaction of known quantities of metal with fixed concentrations of neural mitochondria constituted the test system; oxygen consumption served as the quantitative index of activity. Mitochondria were isolated from homogenates of neural or splenic tissue, prepared by a Blaessig glass hand homogenizer to contain i.o gm of tissue in IO ml 0.2sM 6ucrose. Dense fragments were removed by centrifugal forces of 8oo and I,500 X g, each for IO minutes. Mitochondria were collected, doubly washed in 0.25M sucrose and re-collected by centrifugation at I2,000 X g for 20 minutes. The composition of these mitochondrial fractions was evaluated by phase and electron microscopy. The "test model" was standardized as follows: neural mitochondria were isolated from freshly killed rat brain tissue then suspended in 0.25M sucrose to contain Ioo pg of nitrogen per ml.2 As 0.2 ml aliquots, these were placed in manometric chambers of a Gilson Warburg apparatus together with 2 ml sodium acid maleate buffer at ph 4.5, and enriched with I5o mg per cent glucose.3 The center well contained 0.2 ml of Io per cent KOH absorbed by filter paper. Copper in the form of cupric nitrate, 40 Ag This work was supported in part by United States Public Health Grant NB-o3670 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. Accepted for publication, December 28, i

2 7I4 VOGEL AND KEMPER Vol. 48, No..5 in 0.2 ml distilled-demineralized water, was added from the side arm after a I5- minute equilibration time, to a temperature of 250 C, and followed by 3o minutes of standard running time.4 Readings were taken at io-minute intervals for a period of I50 minutes. Manometric tests were regularly performed in duplicate or triplicate and were often repeated. Into the main chamber of this system was added one of the following: (a) splenic mitochondria, either intact or sonicated; (b) dialysates of splenic mitochondria, before and after precipitation by TCA or perchloric acid; and (c) fractions of splenic dialysate separated by chromatographic columns. The latter were added in amounts relative to their amino acid concentrations 5; the former, in relation to their nitrogen content.2 Customarily, sonication was performed upon pooled preparations of mitochondria from 72 rats suspended in IO ml demineralized water. This suspension was contained in cellulose tubing that in turn was suspended in chilled demineralized water in the chamber of a Raytheon sonic oscillator. Approximately o.875 amperes of current were applied 3 times for 3-minute intervals. Dialysis of the sonicate, diluted to approximately 200 ml with demineralized water, was carried out by means of an LKB 6300 A ultrafilter against a vacuum and at 40 C. The dialysate was collected daily for 5 to 6 days and frozen at - 20 C until a final volume of i6o to i8o ml was obtained. It was then concentrated by a Rotary Evapomix at 370 C to a volume of io ml. This concentrate was precipitated with 5 per cent TCA or 3.3M perchloric acid, added to obtain a final concentration of o.33m. The precipitate was separated by centrifugation at 3,ooo rpm for io minutes, and only the supernate was retained. Regularly, this preparation was strongly acid at a ph of approximately o.8. To rid the supernatant of sucrose, the chelating constituents were first tagged with IOO Mg copper per ml, as cupric nitrate, and the solution was made alkaline to ph 8.o with in NaOH. Aliquots of 2 ml were placed on Dowex AGi - Xio, California Corporation for Biochemical Research, Los Angeles, Calif. (Calbiochem), 200 to 400 mesh, chloride form, in a column i X i5 cm. The column was equilibrated with o.in NaOH. After entry into the bed, each aliquot was washed with 50 ml o.in NaOH, followed by 50 ml demineralized water at neutral ph. Samples were eluted with o.in HCI. The copper marker was detected by spot tests made upon dried filter paper strips that had been previously dipped in o.i per cent rubeanic acid in 70 per cent ethanol. Minute traces of copper yielded an immediate olive green-to-dark mahogany green color. Fractions containing copper were pooled and condensed at 370 C to a final volume of 6 ml. The peptide components of this condensate were then separated by Dowex AG5o- WX2 (Calbiochem) 200 to 400 mesh, hydrogen form, in a i X I5 cm column using 2 ml aliquots. Elution was accomplished with distilled-demineralized water at ph The copper-positive samples, again as detected by spot tests on rubeanic acid paper, were condensed to a final volume of 6 ml. Characteristically, these were notably acid at ph of approximately o.6. Their amino acid content, as quantitated by ninhydrin 5 disclosed concentrations of approximately 5,uM per ml. The composition of these preparations was then characterized by paper chromatography and electrophoresis. Aliquots of io Iul were tagged, either with 2 gg copper, as cupric nitrate, or with copper-64-p (specific activity 65.9 curies per gm from Oak Ridge National Laboratory, Oak Ridge, Tenn.). These were spotted on Whatman filter paper #3 for ascending chromatography at room temperature, in sealed containers, and with running times of 4 hours and 30 minutes. The following buffer was used as a solvent: 70 gm ammonium acetate and 75 ml water, adjusted with concentrated NaOH to ph 7.5, and brought to a final volume of I,000 ml in a 3:7 ratio with ethyl alcohol. Other aliquots of 20 or 40 Ml were spotted upon Beckman filter paper strips (S&S 2,043 A) for electrophoresis in a Beckman Spinco Model "L". The solvent was comprised of IOO ml of pyridine and 4 ml of glacial acetic acid made with

3 May, I966 INTRAMITOCHONDRIAL POLYPEPTIDE 715 water to a volume of i,ooo ml. Constant current of 500 volts was applied for 3 hours at 40 C. The chromatographic and electrophoretic strips were sprayed with either ninhydrin or rubeanic acid (o.i per cent in 70 per cent ethyl alcohol), or were stained with brom-phenol-blue (Beckman dye B-4), or monitored for radioactivity by a Vanguard auto-scanner #880. RESULTS Concentrations of copper that regularly uncoupled oxidative phosphorylation of neural mitochondria, as well as those from the heart and skeletal muscle, did not manifest this property with the mitochondria from the spleen, liver, kidney, or lung of the same rat (Table I). In TABLE I CATALYTIC EFFECTS OF COPPER UPON THE RESPIRATION OF MITOCHONDRIA FROM DIFFERENT ORGANS OF THE SAME RAT 02 consumption (ml) by mitochondrial Source of suspension (ioo /g N) in go minutes mitochondria No Cu Cu (40,ug) Brain 12.7 I59.2 Heart I6.8 1 I3.8 Skeletal muscle I Liver Kidney 5.I 6.5 Spleen 9.1 I3.5 Lung the competitive environment of admixed splenic and neural mitochondria, a constituent of the splenic mitochondria inactivated notable quantities of copper, thus negating the uncoupling action of the metal with the neural mitochondria (Text-fig. i). Sufficient sonication to disrupt the physical integrity of these splenic mitochondria did not quantitatively alter this property. Furthermore, this constituent readily passed through dialysis membranes and resisted precipitation by TCA or perchloric acid (Text-fig. i). When complexed or tagged with copper and fractionated by column chromatography, the copper binding constituent responded chromatographically and electrophoretically as a unit (Figs. i and 2). In the latter instance it moved toward the anode with electrophoretic properties similar to those of copper itself. Upon paper chromatography, the substance was mobile, again as a unit, with an Rf value of o.69. This mobility, though not identical with, was similar to, that of copper, suggesting that the presence of the metallic ion itself is cardinal to the electrophoretic and chromatographic behavior of this molecule. Through its affinity for copper, the peptide was readily tagged and detected by rubeanic acid or radioactivity. By reason of this metallic ion it was rendered ninhydrin-negative f though direct chemical analyses

4 7I VOGEL AND KEMPER Vol. 48, No. 5 showed the purified preparation to contain approximately 5,AM amino acid per ml. Aliquots of this final preparation containing as little as i MM amino acid when added to manometric chambers, together with neural mitochondria having a nitrogen content of I00 M/g, neutralized the uncoupling action of 40 /Ag of copper with the latter (Text-fig. i). Thus the avidity Rat Brain Mitochondria (RBM) + Cu 110-2RBM+H20_ 3 - Rot Spleen Mitochondria (RSM) + Cu 100 4mRaM+Hg 5=RBM+R_ZM+Cu 2F 6a.f "' +H20 z49o o 80-7 a + Rat Spleen Diolysote + Cu i; 8 ạ +0.2ml Polypeptide+Cu E 70 9' A +0.3m1 " co 60_ / 30-40pg Cu or H I TIME, MINUTES TEXT-FIG. i. The effects of 40,ug of copper upon the respiration of rat brain mitochondria and the influence of admixed splenic mitochondria and subfraction therefrom upon this 02 consumption. of the polypeptide for the metal was emphasized by the fact that this appetite denoted binding sites left unsaturated by the copper marker. DIsCUSSION In viewing the conflict of cells with toxins, abundant attention has been paid to the manner of cell injury,7-9 but notably less to the mechanisms of the intracellular resistance. Yet, a cognizance of the latter is essential to lend proper perspective to our understanding of cytotoxic reactions. The present studies underscore the existence of at least two antagonistic intramitochondrial reactions with copper; on the one hand, a toxic action upon oxidative phosphorylation 1; and on the other, the neutralizing effect of chelation. It seems likely that both are common to each mitochondrion, though seemingly in varied equilibrium, for clearly the balance is unfavorably disposed in the neural mitochondria,l while a more salutary reaction is manifested by those of the spleen, liver and kidney. The present studies make it seem likely that in this setting the major determinant of cytotoxicity is the presence or concentration of the chelating polypeptide.

5 May, I966 INTRAMITOCHONDRIAL POLYPEPTIDE 717 The mechanisms of cytotoxicity in hepato-lenticular degeneration (Wilson's disease) have not been clarified. Nevertheless, there is much evidence to indicate that copper plays a cardinal role.'0 With this evidence at hand, one might logically ask why the cells of all tissues are not altered in a degree commensurate with the concentration of the metal. In the normal cell, copper manifests a natural tropism for mitochondria." In Wilson's disease, it has been tentatively suggested that the metal is again selectively concentrated within the mitochondria,l or within the lysosomes.'2 Though each of these organelles has been designated as the prime locus of toxicity-clearly, incrimination by physical associationmeaningful knowledge must await functional studies upon cells and cell fractions from patients with this disorder. It might be pointed out, however, that the role of lysosomes in the degradation of cellular debris places this organelle in a natural position to acquire cellular excesses. But how functionally significant are these accumulations? Ants in the garbage are cause for considerably less concern than those in the bread box. Although the molecular nature of the intramitochondrial polypeptide has not been fully characterized, its properties differ notably from other known naturally occurring biologic substances with affinities for copper. The molecular weight (mol wt), the hydrogen ion concentration, and the electrophoretic mobility distinguish it from cytochrome C oxidase (mol wt 93,ooo) and ceruloplasmin (mol wt 15 I,ooo).13 The lack of color serves to differentiate further the mitochondrial peptide (mitocuprein) from ceruloplasmin and also from erythrocuprein (mol wt 28,000 and hemocuprein (mol wt 35,000).13 The copper protein of the liver (CuLP) has a relatively high molecular weight and has not been specifically associated with the mitochondria,13 while the absence of phenolic oxidase properties serves to distinguish the intramitochondrial peptide from tyrosinase. SUMMARY A polypeptide with avid chelating properties for copper has been identified in the mitochondria of the spleen of the rat. By its chelating properties, it is strongly antagonistic to the uncoupling action of this metal upon oxidative phosphorylation. The findings underscore the advisability of viewing the ecologic struggle of cells with greater awareness of the intrinsic protective mechanisms that may monitor such reactions. REFERENCES I. VOGEL, F. S., and KEMPER, L. Biochemical reactions of copper within neural mitochondria with consideration of the role of the metal in the pathogenesis of Wilson's Disease. Lab. Invest., I963, I2, 17I-I79.

6 7I8 VOGEL AND KEMPER Vol. 48, No KOCH, F. C., and MCMEEKIN, T. L. A new direct nesslerization micro- Kjeldahl method and a modification of the Nessler-Folin reagent for ammonia. J.Amer.Chem. Soc., I924, 46, LONG, C. Biochemists' Handbook. Van Nostrand Co., Inc., New York, N.Y., I96I, P UMBREIT, W. W.; BuRRis, R. H., and STAUFFER, J. F. Manometric Techniques. A Manual describing Methods applicable to the Study of Tissue Metabolism. Burgess Publishing Co., Minneapolis, Minn., I964, ed. 4, 305 pp. 5. ROSEN, H. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys., I957, 67, CRUMPLER, H. R. and DENT, C. E. Distinctive test for a-amino acids in paper chromatography. Nature (London), 1949, I64, 44I PAYLING-WRIGHT, G. Chapter X: The Effects of Injurious Agents upon Cells -Degeneration and Necrosis. In: An Introduction to Pathology. Longmans Green & Co., New York, N.Y., i956, pp. I55-I SMUCKLER, E. A.; ISERI, 0. A., and BENDITT, E. P. Studies on carbon tetrachloride intoxication. II. Depressed amino acid incorporation into mitochondrial protein and cytochrome c. Lab. Invest., I964, 13, 53I FARBER, E. Studies on the chemical pathology of lesions produced by ethionine. Arch. Path. (Chicago), I959, 67, I-8. IO. BEARN, A. G. Wilson's disease; an inborn error of metabolism with multiple manifestations. Amer J. Med., I957, 22, I. PORTER, H., and AINSWORTH, S. The intracellular distribution of copper in brain. J. Neurochem., I96I, 7, I 2. SCHAFFNER, F.; STERNLIEB, I.; BARKA, T., and POPPER, H. Hepatocellular changes in Wilson's disease; histochemical and electron microscopic studies. Amer. J. Path., I962, 4I, 3I I3. WALSHE, J. M., and CUMINGS, J. N. (eds.). Wilson's Disease. Some Current Concepts. Charles C Thomas, Springfield, I96I, p. S. The assistance of Dr. Kenneth McCarty, Department of Biochemistry, Duke University Medical Center, is very gratefully acknowledged. LEGENDS FOR FIGURES FIG. I. Electrophoretic mobility of the splenic mitochondrial polypeptide tagged with 64C. The strip below has been sprayed with rubeanic acid; a scan of its radioactivity is shown above. FIG. 2. A paper chromatograph of the splenic mitochondrial polypeptide tagged with copper and stained with rubeanic acid. The origin (X) is on the left; the solvent line, at the extreme right.

7 --mll", "i afto?", iw.,p-.f. _..-,.'XS. ' -. ' &~~~..,s_'..- May, I966 INTRAMITOCHONDRIAL POLYPEPTIDE 7I v 'i s" -. '.._.. _. -. r - - w..a--wx w._ ^ _ ;'''~~~~~... A-' pg s -; ; :. g v I w ; _. 3O We>s,;i.<; Ft 2

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